Triggering receptor expressed on myeloid cells 2 (TREM2) is an immune modulatory receptor expressed in microglial cells in the brain. Coding variants in TREM2 have been identified as risk factors for Alzheimer's disease (AD). Previous studies have extensively demonstrated that TREM2 plays a central role in microglia activation/survival and amyloid pathology in various cell-based and animal models of AD. Despite evidence demonstrating the functional importance of TREM2 in microglial biology relevant to AD, we currently do not have a complete mechanistic understanding of microglial TREM2 signaling, due in part to the lack of a comprehensive knowledge of TREM2-bearing molecular complex(es) in microglial cells. Most of the focus has been on the interaction of TREM2 with DNAX-activating protein 12 (DAP12), which appears to serve as a communal platform for downstream signaling. Given the complexity and high disease relevance of TREM2-associated biological processes, it is conceivable that additional molecular components may interact with TREM2 and contribute substantially to the regulation of relevant microglial functions. Therefore, we are proposing an unbiased proteomic approach to identify novel components of TREM2-harboring protein complex(es), not yet implicated in TREM2 signaling. Moreover, certain protein-protein-interactions involving TREM2 may be regulated in response to TREM2 receptor stimulation and/or affected by AD-associated TREM2 risk variants. Since conventional protein complex isolation methods are disruptive and often severely affect the stability of the complex, our experimental approach will use an enzyme-catalyzed proximity labeling technique to investigate the protein interactions in living cells. Specifically, the ascorbate peroxidase (APEX2)-based proximity-tagging method combined with mass spectrometry will identify proximal endogenously interacting proteins of TREM2 in the intact microglial cell. Successful completion of the proposed research will fill a critical gap in our understanding of the complex biological regulation of TREM2. Our proposal will establish a protein-protein interaction map of TREM2 in microglial cells and may discover novel TREM2-interacting proteins that are critical for TREM2-mediated microglial functions relevant to AD.